Confocal laser scanning microscope

Purified T‐exos were labelled with PKH67 Fluorescent Cell Linker Kits (MIDI67; Sigma‐Aldrich) and then diluted with PBS and ultracentrifuged at 120 000 g for 70 minutes at 4°C to remove unbound dye. After staining with 10 μmol/L Dil fluorescent cell membrane probe (C1036; Beyotime) for 30 minutes and washing with PBS, 5 × 10⁵/mL Jurkat cells were co‐cultured with PKH‐67 labelled exosomes in confocal dishes. At the end of incubation, cells were washed for three times, fixed with 4% paraformaldehyde for 15 minutes, stained with DAPI and observed under a confocal laser scanning microscope (Olympus Optical Co Ltd.) or measured by FACS Calibur flow cytometry.

The treated chondrocytes were fixed with 1 mL 4% paraformaldehyde for 25 minutes, permeated with 0.2% Triton X-100 in PBS for 5 - 10 min, and blocked with 5% BSA for 90 minutes at room temperature. Then, the chondrocytes were incubated at 4°C for 24 hours with the primary antibody, rabbit polyclonal antibody against Ki67(1:1000, ab15580, Abcam) and mouse monoclonal antibody against Aggrecan (1:1000, ab3778, Abcam). Subsequently, Alexa Fluor594-conjugated (ab150116, Abcam) or Alexa Fluor488-conjugated (ab150077, Abcam) secondary antibody was diluted at a 1:500 with PBS and incubated with the chondrocytes in the dark for 90 minutes. Ultimately, 4',6-Diamidino-2-Phenylindole (DAPI) solution was added for 10 minutes incubation in the dark at ambient temperature. A confocal laser scanning microscope (Olympus Optical Co., Ltd., Tokyo, Japan) was employed to observe the positively stained cells. The fluorescence intensity was assessed by the ImageJ software 2.1.

Setal development was examined using an Olympus Confocal Laser Scanning Microscope (CLSM) and standard compound microscopes. For this, developing pupal tissues of the growing adult epidermis from stages P2–P7 (Tian & Hines, 2018 (link)) were excised from metasomal tergites (spinulate setae) and the mesosomal dorsum (mostly plumose setae) using dissections in cold phosphate buffer solution (PBS) and removal of non-epidermal tissues as much as feasible for the tissue type and stage. These were examined for patterns of setal branching and growth across development. An Olympus BX51 compound microscope with a long working distance 40X objective lens with the sample placed in PBS or glycerol was used for imaging.

A static biofilm growth system was conducted in 96-well plates (Nunclon®, Roskilde, Denmark). Bacterial suspensions with absorbance measurements at 660 nm (A₆₆₀) of between 0.05 and 0.13 were incubated in LB media for 24 hours at 37 °C and 5% CO₂ with vigorous shaking. The biofilm was subjected to two washes with 0.9% saline. Biofilms located at the bottom of the micro-wells were analysed using an Olympus confocal laser scanning microscope (CLSM) with 10 × lenses and 488/510 and 545/610 nm excitation/emission filters. Signals were produced by bacteria harbouring the pMRP9-1 plasmid. GFP produced and localized to live cells was also detected, similar as in previous studies2 21 (link). All signals were calculated using Olympus FLUOVIEW FV300 application software (Tokyo, Japan). The biofilm formed on the sides of microwells at the liquid-air interface and this region was specifically analysed using crystal violet as a control (Supplementary Data Figure S3).

The SkBr3 cells were plated on18-mm cover glasses in DMEM medium and incubated for 24 h so that approximately 70% confluence was reached. The cells were then incubated in the presence or absence of 10 μM HS-133 for 4 h. After washing with phosphate buffered saline (PBS) three times, each slide was covered with DABCO (Sigma-Aldrich) and observed using a confocal laser scanning microscope (Olympus, Tokyo, Japan).

The expression of myofibre‐specific myosin heavy chain (MyHC), which is a morphological parameter of muscle differentiation,²³ was detected in myotubes with immunofluorescence staining. Briefly, the treated C2C12 myotubes were fixed in 4% paraformaldehyde for 15 min, washed with PBS 3 times, incubated with Triton X‐100 and blocked with 5% (v/v) normal goat serum as previously described.²⁴ An anti‐MyHC antibody (1:200 dilution, Abcam) was used for staining, and images were acquired using a confocal laser scanning microscope (Olympus).